The development of recombinant DNA technology has permitted the production of foreign products in host organisms which have been transformed with foreign DNA sequences that encode for those products. In general, the DNA coding for the desired polypeptide or protein is introduced into an appropriate site of a cloning vector in order to form a hybrid molecule which is introduced into a compatible cell. A culture of the cell containing the hybrid DNA molecule is used to express the product encoded by the DNA molecule.
The most extended and known use of the bacterium Xanthomonas campestris pv campestris is the production of xanthan gum, which is a polysaccharide of great commercial value, since this gum in solution is an excellent emulsifier and viscosifier. It is used in the oil industry for enhanced oil recovery. It is also used in agroindustry as well as food and cosmetic industries. The installed world capacity for xanthan gum production is estimated to be 20,000 tons per year. (Yalpani and Sanford. Industrial polysaccharides: Genetic engineering structure/property relations and applications. Elsevier Sciences. Amsterdam, 1989.)
There have been several reports regarding the use of Xanthomonas campestris pv campestris for xanthan gum production and the optimization of this process; for example, Weber and Horan (U.S. Pat. No. 3,271,267) describe a fermentation to obtain xanthan gum using ground cereal as substrate. On the other hand, there have been some reports about the construction of recombinant Xanthomonas campestris strains with the aim of increasing xanthan gum production by overexpressing some of the genes coding for enzymes involved in its biosynthesis. (Thorne et al. 1987. J. Bacteriol. 169: 3593-3600. Harding et al. 1987. J. Bacteriol. 169: 2854-2861).
Genetic engineering technology has also been used to construct Xanthomonas campestris strains with a wider capacity to use growth substrates. In this respect a recombinant strain producing .beta.-galactosidase from transposon Tn 951 (Walsh et al. 1984. Appl. Environ. Microbiol. 47: 253-257), and another expressing .alpha.-amylase from Bacillus (Stripecke et al. 1989 Appl. Microbiol. Biotechnol. 31: 512-517) have been reported.
The bacterium Xanthomonas campestris pv campestris has been defined as GRAS (generally regarded as safe) by the U.S. Food and Drug Administration (FDA) agency; therefore products derived from this bacterium would have less restrictions on use in food or pharmaceutical products than the same products expressed by other Gram-negative bacteria such as Escherichia coli or Pseudomonas aeruginosa.
Research carried out at the Western Utilization Research Laboratory and at Woodward Research Corporation with respect to xanthan gum toxicity as a supplement of rats and dog food, showed that it is resistant to degradation by the digestive system and therefore its use as food additive was approved by FDA.
The most widely used bacterium for the expression of heterologous proteins has been Escherichia coli. Several lipases derived from Pseudomonas have been expressed using E. coil as host (Othera et al. Japanese patent laid open 60-188072, Kugimiga et al. Japanese patent laid open 62-228279, Wolfare and Winkler. European patent no. 0334462 A1). One of the principal limitations of this host is that enzymes are not secreted so the purification of the recombinant products is more difficult and expensive. Xanthomonas campestris pv campestris natural isolates produce extracellular enzymes such as proteases and carbohydrases (Daniels, 1989. In Hopwood and Chater eds. Genetics of bacterial diversity. Academic Press.). This characteristic permits the production of extracellular heterologous proteins using Xanthomonas campestris pv capastri as a host organism for recombinant DNA molecules.
Some Gram-negative bacteria, besides Xanthomonas campestris, are able to produce extracellular proteins. These bacteria include Erwinia, Pseudomonas and some Klebsiella species. The protein secretory machinery present in all these Gram-negative bacteria has important similarities (Filloux et al. 1990. EMBO J. 9: 4323-4329).
Some Pseudomonas strains have been used as hosts to express heterologous extracellular lipases derived from different Pseudomonas species (Nakanishi et al. European patent No. 0331 376 A2).
A general characteristic of proteins is that they are stabilized in solution in the presence of viscosifying agents such as glycerol or polyethyleneglycol. In this respect, the activity of a Pseudomonas aeruginosa lipase has been reported to be enhanced by the presence of certain polysaccharides such as cellulose or hyaluronate (Jager and Winkler 1983. Fems Microbiol. Lett. 19: 59-63).
The present invention refers to a process of obtaining extracellular recombinant products using Xanthomonas campestris pv campestris as host, in such a way that the stability of said extracellular recombinant product is enhanced by the presence of xanthan gum. The advantages of the present invention are the following:
The simplicity to purifying the recombinant product and the xanthan gum which are produced extracellularly by Xanthomonas campestris pv campestris.
The possibility of having less restrictions to introduce in food or pharmaceutical products the recombinant products produced by Xanthomonas campestris pv campestris, and
The enhanced stability of the extracellular recombination heterologous proteins since they are produced in the presence of xanthan gum.